The transformation of a non-malignant human breast epithelial cell to a malignant state is accompanied by the alteration of a specific protein, namely proliferating cell nuclear antigen (PCNA). It arises not because of a genetic mutation but through post-translational modification of the protein. To date, there is an exact correlation between an observed decrease in DNA synthesis fidelity and the expression of the cancer specific isoform of PCNA (csPCNA) in malignant breast cells and tissue. We recently developed a novel antibody that specifically detects only the csPCNA isoform, this antibody has enabled us to selectively identify the csPCNA isoform in breast cancer cells and tumor tissues. Overall, our studies suggest the csPCNA isoform uniquely expressed in malignant cells may be a bona fide hallmark of cancer and as such must, therefore, play an important role in the life of cancer cells. This evidence indicates that a thorough structure and function analysis of csPCNA in breast cancer cells may lead to important new insights into its role in cancer cell proliferation and progression. We propose to: (1) identify the post-translational modification(s) responsible for the expression of csPCNA polypeptide in breast cancer cells, (2) determine the role csPCNA plays in the aberrant DNA synthesis fidelity of breast cancer cells, (3) evaluate DNA synthesis fidelity and csPCNA levels during breast cancer progression, (4) determine whether csPCNA displays differential interactions with proteins that have been identified as PCNA binding partners, and (5) validate that the presence of csPCNA in breast tissue is a true indicator of malignancy. Our discovery of csPCNA points to a novel mechanism whereby the alteration of a specific type of post-translational modification may play a key role in abrogating cell cycle check points and contributing to the cascade of events which lead to the accumulation of genetic damage sustained by the cancer cell and ultimately the transformation of the normal cell. Also, the recent development of an antibody that specifically recognizes csPCNA holds the real potential for developing a highly selective reagent that may significantly improve the early detection of malignant breast cells, improve monitoring of remission status, and potentially save thousands of lives each year. In addition, the dissection of its role in reducing DNA synthetic fidelity in malignant cells could suggest potential new targets for therapeutic development.